The long term goal of this proposal is to understand the role of Mg2+ in cellular physiology. Mg2+ is by far the most abundant intracellular divalent cation, its intracellular concentration is tightly controlled, it is required for the integrity of cell structural components, and it is necessary for the activity of many important metabolic enzymes. Moreover, recent work from this and other laboratories has suggested that Mg2+ plays a fundamental regulatory role in prokaryotic and eukaryotic cells. It seems probable therefore that all cells possess interesting mechanisms for sensing and controlling the entry and exit of Mg2+ through the cell membrane. The preliminary data presented establish the existence of three loci (corA, mgtA, and mgtB) in Salmonella typhimurium which appear to represent three distinct mg2+ transport systems. At least two other loci, corB and corC, have been identified that interact with the CorA transport system. Transport by the MgtA and MgtB systems is regulated by the extracellular concentration of Mg2+ across the cell membrane in S. typhimurium. Characteristics of individual transport systems will be determined in mutant strains carrying only one functional Mg2+ transport system. Aim I proposes a molecular analysis of the genes and gene products involved in Mg2+ transport through sequencing of the transport genes already identified and cloned and the determination of the sites of initiation and termination of transcription. Aim II will extend the genetic analysis of Mg2+ transport already begun by seeking additional mutations that affect function of each of the three transport systems. Any such new mutations will be localized on the chromosome, their phenotypic properties investigated, and their molecular properties determined as described in Aim I. Aim III will characterize the properties of the individual Mg2+ transport systems. Studies will include investigation of a) the cation specificity and kinetic parameters of cation transport of each system, b) the properties of Mg2+ efflux which is mediated solely by the CorA transport system, c) the source of energy for each transport system, using inhibitor studies in unc mutants which defective in energy coupling, and d) lacZ operon fusions to the promoters of mgtA and mgtB to characterize the phenomenology of Mg2+ regulation of these loci.